Hrp linked anti mouse and rabbit igg antibodies

After pretreatment, OGD injury, and restoration, cells were washed rapidly with ice-cold PBS, scraped, and collected. Cell pellets were lysed with ice-cold RIPA buffer (Sigma, MO, USA). The lysates were centrifuged at 13,200 rpm for 1 h at 4°C to produce whole-cell extracts. Protein content was quantified using the BCA method (Pierce, IL, USA). Protein (20 μg) was separated on a 10% SDS-polyacrylamide (PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin, prepared in Tris-buffered saline/Tween (TBS-T; 20 nM Tris (pH 7.2); 150 mM NaCl; 0.1% Tween 20), for 1 h at RT, immunoblots were incubated overnight at 4°C with primary antibodies that specifically detect Akt (1 : 2000, Cell Signaling, MA, USA), p-Akt (1 : 2000, Cell Signaling, MA, USA), JNK (1 : 2000, Cell Signaling, MA, USA), p-JNK (1 : 2000, Cell Signaling, MA, USA), Claudin 5 (1 : 1000, Santa Cruz, CA, USA), VEGF (1 : 1000, Millipore, MA, USA), Bax (1 : 2000, Cell Signaling, MA, USA), or β-actin (1 : 2000, Cell Signaling, MA, USA). Next, blots were incubated with HRP-linked anti-mouse and -rabbit IgG antibodies purchased from Abcam (Cambridge, MA, USA) for 1 h at RT. Enhanced chemiluminescence was performed by ECL (Pierce, IL, USA) [54 (link)].